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The maintenance of highly proliferative capacity and full differentiation potential is a necessary step in the initiation of stem cell-based regenerative medicine. Our recent study showed that epidermal growth factor (EGF) significantly enhanced hair follicle-derived mesenchymal stem cell (HF-MSC) proliferation while maintaining the multilineage differentiation potentials. However, the underlying mechanism remains unclear. Herein, we investigated the role of EGF in HF-MSC proliferation. HF-MSCs were isolated and cultured with or without EGF. Immunofluorescence staining, flow cytometry, cytochemistry, and western blotting were used to assess proliferation, cell signaling pathways related to the EGF receptor (EGFR), and cell cycle progression. HF-MSCs exhibited surface markers of mesenchymal stem cells and displayed trilineage differentiation potentials toward adipocytes, chondrocytes, and osteoblasts. EGF significantly increased HF-MSC proliferation as well as EGFR, ERK1/2, and AKT phosphorylation (p-EGFR, p-ERK1/2, and p-AKT) in a time- and dose-dependent manner, but not STAT3 phosphorylation. EGFR inhibitor (AG1478), PI3K-AKT inhibitor (LY294002), ERK inhibitor (U0126), and STAT3 inhibitor (STA-21) significantly blocked EGF-induced HF-MSC proliferation. Moreover, AG1478, LY294002, and U0126 significantly decreased p-EGFR, p-AKT, and p-ERK1/2 expression. EGF shifted HF-MSCs at the G1 phase to the S and G2 phase. Concomitantly, cyclinD1, phosphorylated Rb, and E2F1expression increased, while that of p16 decreased. In conclusion, EGF induces HF-MSC proliferation through the EGFR/ERK and AKT pathways, but not through STAT-3. The G1/S transition was stimulated by upregulation of cyclinD1 and inhibition of p16 expression.

Background Cytoplasmic mislocalization and aggregation of TAR DNA-binding protein-43 (TDP-43) is a hallmark of the amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) disease spectrum, causing both nuclear loss-of-function and cytoplasmic toxic gain-of-function phenotypes. While TDP-43 proteinopathy has been associated with defects in nucleocytoplasmic transport, this process is still poorly understood. Here we study the role of karyopherin-β1 (KPNB1) and other nuclear import receptors in regulating TDP-43 pathology. Methods We used immunostaining, immunoprecipitation, biochemical and toxicity assays in cell lines, primary neuron and organotypic mouse brain slice cultures, to determine the impact of KPNB1 on the solubility, localization, and toxicity of pathological TDP-43 constructs. Postmortem patient brain and spinal cord tissue was stained to assess KPNB1 colocalization with TDP-43 inclusions. Turbidity assays were employed to study the dissolution and prevention of aggregation of recombinant TDP-43 fibrils in vitro. Fly models of TDP-43 proteinopathy were used to determine the effect of KPNB1 on their neurodegenerative phenotype in vivo. Results We discovered that several members of the nuclear import receptor protein family can reduce the formation of pathological TDP-43 aggregates. Using KPNB1 as a model, we found that its activity depends on the prion-like C-terminal region of TDP-43, which mediates the co-aggregation with phenylalanine and glycine-rich nucleoporins (FG-Nups) such as Nup62. KPNB1 is recruited into these co-aggregates where it acts as a molecular chaperone that reverses aberrant phase transition of Nup62 and TDP-43. These findings are supported by the discovery that Nup62 and KPNB1 are also sequestered into pathological TDP-43 aggregates in ALS/FTD postmortem CNS tissue, and by the identification of the fly ortholog of KPNB1 as a strong protective modifier in Drosophila models of TDP-43 proteinopathy. Our results show that KPNB1 can rescue all hallmarks of TDP-43 pathology, by restoring its solubility and nuclear localization, and reducing neurodegeneration in cellular and animal models of ALS/FTD. Conclusion Our findings suggest a novel NLS-independent mechanism where, analogous to its canonical role in dissolving the diffusion barrier formed by FG-Nups in the nuclear pore, KPNB1 is recruited into TDP-43/FG-Nup co-aggregates present in TDP-43 proteinopathies and therapeutically reverses their deleterious phase transition and mislocalization, mitigating neurodegeneration. Graphical Abstract

IntroductionSuccessful stem cell therapy relies on large-scale generation of stem cells and their maintenance in a proliferative multipotent state. This study aimed to establish a three-dimension culture system for large-scale generation of hWJ-MSC and investigated the self-renewal activity, genomic stability and multi-lineage differentiation potential of such hWJ-MSC in enhancing skin wound healing.MethodshWJ-MSC were seeded on gelatin microbeads and cultured in spinning bottles (3D). Cell proliferation, karyotype analysis, surface marker expression, multipotent differentiation (adipogenic, chondrogenic, and osteogenic potentials), and expression of core transcription factors (OCT4, SOX2, NANOG, and C-MYC), as well as their efficacy in accelerating skin wound healing, were investigated and compared with those of hWJ-MSC derived from plate cultres (2D), using in vivo and in vitro experiments.ResultshWJ-MSC attached to and proliferated on gelatin microbeads in 3D cultures reaching a maximum of 1.1–1.30 × 107cells on 0.5 g of microbeads by days 8–14; in contrast, hWJ-MSC derived from 2D cultures reached a maximum of 6.5 -11.5 × 105 cells per well in a 24-well plate by days 6–10. hWJ-MSC derived by 3D culture incorporated significantly more EdU (P < 0.05) and had a significantly higher proliferation index (P < 0.05) than those derived from 2D culture. Immunofluorescence staining, real-time PCR, flow cytometry analysis, and multipotency assays showed that hWJ-MSC derived from 3D culture retained MSC surface markers and multipotency potential similar to 2D culture-derived cells. 3D culture-derived hWJ-MSC also retained the expression of core transcription factors at levels comparable to their 2D culture counterparts. Direct injection of hWJ-MSC derived from 3D or 2D cultures into animals exhibited similar efficacy in enhancing skin wound healing.ConclusionsThus, hWJ-MSC can be expanded markedly in gelatin microbeads, while retaining MSC surface marker expression, multipotent differential potential, and expression of core transcription factors. These cells also efficiently enhanced skin wound healing in vivo, in a manner comparable to that of hWJ-MSC obtained from 2D culture.

Cell therapy and cell-based tissue engineering is becoming increasingly important in regenerative medicine. Stem cells that are characterized by self-renewal, high proliferation and multiple differentiation potentials have attracted attention in cell-based regenerative medicine. Maintaining the aforementioned characteristics of stem cells is the first key step in cell-based regenerative medicine. Basic fibroblast growth factor (bFGF) is a well-known growth factor that efficiently maintains the self-renewal, high proliferation and multilineage differentiation potential of stem cells. Whether or not other growth factors, such as acidic fibroblast growth factor (aFGF) and epidermal growth factor (EGF) have similar effects has yet to be fully elucidated. Human hair follicle-derived mesenchymal stem cells (HF-MSCs) were obtained by organ culture. They exhibited surface markers of bone marrow mesenchymal stem cells as shown by positive staining for CD44, CD73, CD90 and CD105, and they also displayed trilineage differentiation potentials into adipocytes, chondrocytes and osteoblasts by cytochemistry and qRT-PCR. Flow cytometry analysis showed that up to 70% of HF-MSCs cultured in the presence of aFGF, bFGF or EGF stayed at the G0/G1 phase. Proliferation analysis showed that both bFGF and EGF at as low as 1 ng/ml and aFGF at above 5 ng/ml levels significantly increased the proliferation of HF-MSCs by cell counting. Consistent with proliferation analysis, immunofluorescence staining showed that more than 95% of HF-MSCs cultured in the presence of aFGF, bFGF and EGF were positively stained for proliferating cell nuclear antigen. HF-MSCs cultured in the presence of aFGF, bFGF or EGF retained marked trilineage differentiation potentials. By contrast, HF-MSCs cultured in the absence of bFGF, aFGF and EGF lost multipotency.

Stem cells derived from elderly donors or harvested by repeated subculture exhibit a marked decrease in proliferative capacity and multipotency, which not only compromises their therapeutic potential but also raises safety concerns for regenerative medicine. NANOG—a well-known core transcription factor—plays an important role in maintaining the self-renewal and pluripotency of stem cells. Unfortunately, the mechanism that NANOG delays mesenchymal stem cell (MSC) senescence is not well-known until now. In our study, we showed that both ectopic NANOG expression and PBX1 overexpression (i) significantly upregulated phosphorylated AKT (p-AKT) and PARP1; (ii) promoted cell proliferation, cell cycle progression, and osteogenesis; (iii) reduced the number of senescence-associated-β-galactosidase- (SA-β-gal-) positive cells; and (iv) downregulated the expression of p16, p53, and p21. Western blotting and dual-luciferase activity assays showed that ectopic NANOG expression significantly upregulated PBX1 expression and increased PBX1 promoter activity. In contrast, PBX1 knockdown by RNA interference in hair follicle- (HF-) derived MSCs that were ectopically expressing NANOG resulted in the significant downregulation of p-AKT and the upregulation of p16 and p21. Moreover, blocking AKT with the PI3K/AKT inhibitor LY294002 or knocking down AKT via RNA interference significantly decreased PBX1 expression, while increasing p16 and p21 expression and the number of SA-β-gal-positive cells. In conclusion, our findings show that NANOG delays HF-MSC senescence by upregulating PBX1 and activating AKT signaling and that a feedback loop likely exists between PBX1 and AKT signaling.

The use of human mesenchymal stem cells (hMSCs) in cell therapies has increased the demand for strategies that allow efficient cell scale-up. Preliminary data on the three-dimensional (3D) spinner culture describing the potential use of microcarriers for hMSCs culture scale-up have been reported. We exploited a rich source of autologous stem cells (human hair follicle) and demonstrated the robust in vitro long-term expansion of human hair follicle-derived mesenchymal stem cells (hHF-MSCs) by using CultiSpher(®)-G microcarriers. We analyzed the feasibility of 3D culture by using hHF-MSCs/CultiSpher(®)-G microcarrier constructs for its potential applicability in regenerative medicine by comparatively analyzing the performance of hHF-MSCs adhered to the CultiSpher(®)-G microspheres in 3D spinner culture and those grown on the gelatin-coated plastic dishes (2D culture), using various assays. We showed that the hHF-MSCs seeded at various densities quickly adhered to and proliferated well on the microspheres, thus generating at least hundreds of millions of hHF-MSCs on 1 g of CultiSpher(®)-G within 12 days. This resulted in a cumulative cell expansion of greater than 26-fold. Notably, the maximum and average proliferation rates in 3D culture were significantly greater than that of the 2D culture. However, the hHF-MSCs from both the cultures retained surface marker and nestin expression, proliferation capacity and differentiation potentials toward adipocytes, osteoblasts and smooth muscle cells and showed no significant differences as evidenced by Edu incorporation, cell cycle, colony formation, apoptosis, biochemical quantification and qPCR assays.

Genetically engineered stem cells that overexpress genes encoding therapeutic products can be exploited to correct metabolic disorders by repairing and regenerating diseased organs or restoring their function. Hair follicles are readily accessible and serve as a rich source of autologous stem cells for cell-based gene therapy. Here we isolated mesenchymal stem cells from human hair follicles (HF-MSCs) and engineered them to overexpress the human insulin gene and release human insulin in a time-and dose-dependent manner in response to rapamycin. The engineered HF-MSCs retained their characteristic cell surface markers and retained their potential to differentiate into adipocytes and osteoblasts. When mice with streptozotocin-induced type 1 diabetes were engrafted with these engineered HF-MSCs, these cells expressed and released a dose of human insulin, dramatically reversed hyperglycemia, and significantly reduced death rate. Moreover, the engineered HF-MSCs did not form detectable tumors throughout the 120-day animal tests in our experiment. Our results show that HF-MSCs can be used to safely and efficiently express therapeutic transgenes and therefore show promise for cell-based gene therapy of human disease.